7-Hydroxy chromones as potent antioxidants

ABSTRACT

The present invention describes the identification and purification of 7-hydroxychromes that exhibit potent antioxidant activity. In one embodiment the present invention includes a method for providing an antioxidant to a host in need thereof, comprising administering an effective amount of a 7-hydroxychrome or a mixture of 7-hydroxychromones. The present invention includes methods that are effective in inhibiting free radical and oxidation caused damage through the simultaneous suppression of free radical generation and the suppression of the production of reactive oxygen species (ROS). The present invention also includes methods for preventing and treating ROS mediated diseases and conditions and diseases and conditions associated with other oxidative processes. The method for preventing and treating ROS mediated diseases and conditions and diseases and conditions associated with other oxidative processes is comprised of administering to a host in need thereof an effective amount of a composition comprised of a 7-hydroxychrome or a mixture of 7-hydroxychromones and a pharmaceutically acceptable carrier. Included in this invention is an improved method to isolate and purify 7-hydroxychromones from plant sources.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of antioxidants.Specifically, this invention relates to the identification of a class ofcompounds, referred to herein as 7-hydroxychromones, which have potentantioxidant activity. More specifically, this invention relates to amethod for the prevention and treatment diseases and conditionsassociated with reactive oxygen species (ROS) damage and other oxidationprocesses by administration of one or more 7-hydroxychromones. Includedin this invention is an improved method to isolate and purify7-hydroxychromones from plant sources.

BACKGROUND OF THE INVENTION

[0002] Reactive oxygen species (ROS) are produced by normalphysiological processes in the body and play a significant role in theflow energy and information in all the living systems. (Voeikov (2001)Riv. Biol. 94:237-258). ROS also perform critical functions related tointercellular induction of apoptosis (Bauer (2000) Anticancer Res.20:4115-4139), inflammation processes and immune responses. ROS are alsogenerated as by-products of normal metabolic processes, from foodadditives, from environmental sources, such as ultraviolet radiation(Wenk et al. (2001) Curr. Probl. Dermatol. 29:83-94) and tobacco smoke(Stich et al. (1991) American Journal of Clinical Nutrition53:298S-304S), and from many other pollutants. Reactive oxygen species(ROS) include oxygen related free radicals, such as superoxide (O₂^(−.)), peroxyl (ROO^(−.)), alkoxyl (RO^(−.)), hydroxyl (HO^(−.)), andnitric oxide (NO^(−.)); and non-radical species, such as the singletoxygen (¹O₂), hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl).

[0003] Cells have specific mechanisms to maintain homeostasis, whichkeep ROS levels in check. (Mates (2000) Toxicology 153:83-104). A numberof different biological pathways are employed to maintain oxidativehomeostasis within a cell. These pathways include the synthesis andrecycling of γ-glutamyl-cysteinyl-glycine (Glutathion GSH) and theaction of specific enzymes, such as SOD, catalases and peroxidases.(Deneke et al. (1989) Am. J. Physiol. 257: L163-L173). The generationand recycling of GSH is commonly known as the γ-glutamyl cycle.(Lieberman et al. (1995) Amer. J. Pathol. 147:1175-1185). This cycle,which is illustrated in FIG. 1, culminates in the production of thenaturally occurring intracellular anti-oxidant, GSH. The maintenance ofappropriate levels of GSH is very important to the redox state of thecell.

[0004] Superoxide anions are among the most reactive and damaging ROSproduced by the mitochondria. Consequently, the regulation of theirproduction and neutralization is a very important component ofmaintaining the cellular redox state. The enzyme superoxide dismutase(SOD) catalyzes the production of the less reactive hydrogen peroxidefrom superoxide anions, as illustrated by equation 1. The hydrogenperoxide produced is subsequently reduced to water by either catalase orglutathione peroxidase (GPx). (Wei et al. (2001) Chin. J. Physiol.31:1-11). $\begin{matrix}{{2O_{2^{\cdot}}^{-}}\underset{S\quad O\quad D}{\overset{2\quad H^{+}}{\rightarrow}}{{H_{2}O_{2}} + O_{2}}} & (1)\end{matrix}$

[0005] If the processes that maintain oxidative homeostasis in the cellget out of balance, free radical levels become dangerous, as these arehighly reactive, molecules that damage DNA, proteins and components oncell membranes, eventually leading to cellular damage throughout thebody and contributing a primary role in the aging process.

[0006] As a result of our modern diet and lifestyle, exposure to freeradicals is increasing dramatically having a profound effect on ourvulnerability to disease. The role of oxidative stress and itsassociated age related diseases is well established. Physiologicalchanges that occur as we age, result in the loss of a homeostaticbalance between the generation of ROS, which cause oxidative damage andthe production of naturally occurring antioxidants, such as glutathione(GSH) and other regulatory enzymes (superoxide dismutase, catalase andperoxidases). As the origin of these ROS is the mitochondria, this lossof homeostasis that occurs during aging is known as the “mitochondrialtheory of aging” (Simon (2000) Annals New York Acad. Sci. 908:219-225).Characteristic changes within the mitochondria during aging include, adecrease in the expression of the enzymes Cu/Zn-superoxide dismutase(SOD), which are responsible for “neutralizing” highly reactive andoxidative superoxide anions, accumulation of hydrogen peroxide and thereduction of mitochondrial glutathione pools. There is also a loss ofintracellular and plasma GSH levels, resulting in an increasingly globaloxidative environment within the human body during aging. Thisenvironment translates to a number of problems on both the cellularlevel and the level of the organism itself, as evidenced by thegeneration of chronic diseases of aging associated with oxidativedamage.

[0007] For example, decreased GSH levels have been found in peoplesuffering from debilitating neuro-degenerative diseases such asAlzheimer's Disease, Parkinson's disease, and amyotrophic lateralsclerosis (ALS). Cellular changes within individual cells include,physical damage to cellular components, as well as, alterations incellular responses resulting from the increasingly oxidativeenvironment. The physical damage includes, the oxidative damage to bothcellular and mitochondrial DNA and the peroxidation of lipids within thecellular and mitochondrial membranes. These changes affect the integrityof both of these components and translate to impaired function. Theoxidative environment also contributes to changes in many generesponses. (Forsberg et al. (2001) Arch. Biochem. Biophys. 389: 84-93).This is because transcription factors, such as NF-kB and AP-1 arecontrolled by changes in the redox state of cells. For example, NF-kBtranscription factors are activated in oxidative environments. Thus,minute alterations in the redox state that occur during aging coulddramatically change the way a cell responds to a particular stimulus.The altered responses can manifest themselves as increased apoptosis,cancers or the loss of function that ultimately cause many of thediseases related to aging. (Zs.-Nagy (2001) Annals New York Acad. Sci.928:187-199).

[0008] ROS also a play critical role after brain injury, implicatingthem in the pathology of traumatic CNS damage and cerebral ischemia.(Lewen (2000), J. Neurotrauma 17:871-890). ROS caused oxidative stressin endothelial cells is a leading condition in the pathogenesis of manycardiovascular diseases (Touyz (2000) Curr. Hypertens. Rep. 2:98-105),pulmonary diseases (Berry et al. (2001) Curr. Opin. Nephrol. Hypertens.10:247-255) and metabolic disorders (Takahashi (2000) Nippon Rinsho58:1592-1597).

[0009] Protection of alveolar epithelial cells and vascular endotheliacells against pulmonary (Muzykantov (2001) Antioxid. Redox Signal 3:39-62) and vascular endothelium oxidative stress (Muzykantov (2001) J.Control Release 12:1-21) has been investigated via deliveryantioxidative enzymes, such as SOD and catalase. The beneficial effectsfrom dietary antioxidants, such as resveratrols (Hung et al. (2002) Br.J. Pharmacol. 135:1627-1633) and alpha-lipoic acid (Takaoka et al.(2002) Clin. Exp. Pharmacol. 29:189-194) in reducing the incidence ofcoronary heart diseases, butylated hydroxytoluene and β-carotene inphotocarcinogenesis (Black (2002) Front Biosci. 7:D1044-1055) has beendocumented. Even though antioxidants may reduce free radicals generatedby radiotherapy and chemotherapy, there is no evidence suggesting thatthey interfere with conventional cancer therapy. Clinical evidenceindicates that cancer patients given antioxidants exhibit highertolerance and decreased side effects, resulting from treatment andfurther that they live longer and have a higher quality of life. (Lamsonet al. (1999) Altern. Med. Rev. 4:304-329). While these diseasesrepresent extreme examples, it is well documented that the skewing ofthe redox state toward an oxidative environment is a characteristic ofthe aging process. Therefore, the average person benefits by maintaininga homeostatic oxidative balance. Because the oxidative damage thatoccurs during the aging process can be directly linked to pathologicalaspects of these diseases, controlling or restoring the homeostaticbalance of the oxidative state is of great interest to the medicalindustry as a whole. Consequently, anti-oxidants have a secure placewithin the Dietary Supplemental Health and Education Act (DSHEA) arena,as evidenced by the number of antioxidant products targeted for theanti-aging market.

[0010] Antioxidant defense mechanisms are species specific and heavilyinfluenced by nutrition, since important antioxidants, such as, ascorbicacid and α-tocopherol cannot be synthesized by humans and therefore,must be obtained from ones diet. (Benzie (2000) Eur. J. Nutr. 39:53-61).Antioxidants are very popular dietary supplements in the nutritional andcosmeceutical industries. Types of products promoted as antioxidantsinclude, vitamins (i.e., Vc, Ve, Vb, β-carotene), minerals (i.e.,selenium), amino acids (i.e., lysine, cysteine, n-acetyl cysteine,lipoic acid), phenolic acids (i.e., curcumin, rosveratrol, chetechins,EGCG), flavanoids (i.e., rutin, quercetine, etc.) (Pietta (2000) J. Nat.Prod. 63:1035-1042), anthrocynadines, pycnogenol, coumarine derivatives,polyphenols (i.e., tannins) and many different types of botanicalextracts. Antioxidant products include polyunsaturated fatty acids(PUFAs) and specialty amino acids, claims for which range from reducingthe risk of heart disease to treating joint problems and easingdepression. β-carotene is a major antioxidant, which has been shown toreduce the risk of prostate cancer. β-carotene is primarily available indietary supplements, specifically multivitamin formulations andsingle-entity soft gel capsules. The product is a popular antioxidant,thought to help prevent many diseases.

[0011] Lycopene, an up-and-coming phytochemical in the β-carotenefamily, is receiving increased attention because of its promise as anantioxidant. The new product, Lycopene 5% TG, is readily available inmultivitamin, antioxidant, straight and chewable formulations. SabinsaCorp. has developed a new, colorless tumeric root extract,tetrahydrocurcuminoids (THC), for use in dietary supplements andcosmeceuticals as a bioprotectant and multipurpose antioxidant that doesnot stain. THC is a free radical scavenger, preventing free radicalchain reactions by neutralizing existing free radicals and/ormaintaining a reducing environment around the cells and preventing theformation of free radicals; and acting as a chelating agent to generatenon-active complexes with prooxidatant metals. It is claimed that someantioxidants enhance the protective capability of the cell wall, therebybolstering the cell's defense against free radicals and repairing damagedone to cells by free radicals. However, there is not muchscientifically sound data to prove the efficacy of these antioxidants.

[0012] The Oxygen Radical Absorption Capacity (ORAC) assay is a methodfor measuring total serum antioxidant activity. (Cao et al. (1993) FreeRad. Biol. Med. 14:303-311). It can be used to quantitatively measurethe total antioxidant capacity, as well as, qualitatively measure thelevels of fast versus slow acting antioxidants in a blood serum sample.The assay utilizes the free radical sensitive fluorescent indicatorprotein β-phycoerythrin (β-PE) to monitor the effectiveness of variousserum antioxidants in protecting β-PE from becoming damaged by freeradicals. Assay results are quantitated by allowing the reaction toreach completion and then integrating the area under the kinetic curverelative to a blank reaction containing no antioxidant. The area underthe curve is proportional to the concentration of all the antioxidantspresent in the sample. (DeLang and Glazer (1989) Analyt. Biochem.177:300-306).

[0013] The Lipid Peroxidation Inhibition Capacity (LPIC) assay is amethod for measuring the ability of a sample to inhibit the initiationand propagation of a spontaneous lipid peroxidation reaction. Lipidperoxidation represents the major mechanism of lipid destructionoccurring in an organism. This reaction is also an important source ofreactive oxygen species. In part, lipid peroxidation is controlled invivo by:

[0014] 1) the chelation of trace metals, such as iron and copper, thatare involved in the initiation of lipid peroxidation reactions; and

[0015] 2) the presence of antioxidants that terminate the propagationreaction once it is initiated.

[0016] Lipid peroxidation is believed to be one of the major destructivereactions, occurring in the plasma components and blood vessel walls,leading the onset of cardiovascular disease. (Riemersma et al. (1991)Lancet 337:1-5). Thus, a principal role of antioxidants andmetal-chelating components in the serum is the protection of the entirecardiovascular system through the control of lipid peroxidation.(Stampfer et al. (1993) New England Journal of Medicine 328:1444-1449;Rimm et al. (1993) New England Journal of Medicine 328:1450-1456.).Metal chelators, such as ferritin for iron and metallothionein forcopper are well known, but other serum constituents, such as urate,which can chelate iron, may also be important. (Maples et al. (1988) J.Biol. Med. 263:1709-1712). Antioxidants, such as α-tocopherol, are knownto prevent propagation of lipid peroxidation reactions, but there may bemany other serum constituents that could be equally or even moreimportant than α-tocopherol. (Stahelin et al. (1984) Journal of theNational Cancer Institute 73:1463-1468). The LPIC assay measures howwell a sample is able to both inhibit and terminate a lipid peroxidationreaction. The LPIC value complements the ORAC value. The ORAC assayprovides information on total antioxidant capacity, whereas the LPICassay provides information on both total antioxidant capacity and on thestatus of metal catalysts in effectively protecting against in vivolipid peroxidation reactions.

[0017] Chromones are a specific type of aromatic compounds having abenzopyran-4-one as their major skeletal structure as illustrated by thefollowing general structure:

[0018] wherein

[0019] R₁, R₂ and R₃ are independently selected from the groupconsisting of —H, —OH, —CH₃, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl,hydroxylalkyl, hydroxylalkenyl, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH,—NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, including,but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoylesters, trihydroxybenzoyl esters and caffeoyl esters; thereof carbon,oxygen, nitrogen or sulfur glycoside of a single or a combination ofmultiple sugars including, but not limited to, aldopentoses, methylaldopentose, aldohexoses, ketohexose and their chemical derivativesthereof; dimer, trimer and other polymerized chromones;

[0020] wherein said alkyl and/or alkenyl group is a straight and/orbranched chain having between 1-20 carbon atoms with and/or withoutdouble bonds in different positions;

[0021] X is selected from the group of pharmaceutically acceptablecounter anions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.; and

[0022] R is an alkyl group having between 1-20 carbon atoms.

[0023] To date there are only 183 chromones isolated from naturalsources. (The Combined Chemical Dictionary, Chapman & Hall/CRC, Version5:1 June 2001).

[0024] Chromones reportedly exhibit monoamine oxidase inhibitoryactivity (Fujimoto et al. (2002) Chem. Pharm. Bull. 50:330-336),tyrosinase inhibitory activity (Oiao et al. (2002) Chem. Pharm. Bull.50:309-311), anti-platelet effects (Leoncini et al. (1991) Pharmacol.Res. 23:139-148), inhibitory activity of phosphatidylinositol-3-kinase(Pong et al. (1998) J. Neurochem. 71:1912-1919; Blommaart et al. (1997)Eur. J. Biochem 23:240-246), growth inhibitory activity against oralpathogens (Cai (1996) J. Nat. Prod. 59:987-990), prostagladin H synthaseinhibitory activity (Jurenka et al. (1989) Comp. Biochem. 93:253-255).Chromones also possess therapeutic efficacy against type IIcollagen-induced arthritis in rats (Inaba et al. (2000) Chem. Pharm.Bull. 48:131-139) and hypolipidemic activity (Witiak et al. (1975) J.Med. Chem. 18:935-942; Tetko et al. (1995) Bioorg Khim. 21:809-815). Ithas also been reported that chromones can function as selective sigmareceptor ligands (Erickson et al. (1992) J. Med. Chem. 35:1526-1535).Based on animal studies, chromones are easily absorbed and metabolized(Crew et al. (1976) Xenobiotica 6:89-100) and the c-glucosyl bond ofaloesin can be cleaved by human intestinal bacteria. (Che et al. (1991)Chem. Pharm. Bull. 39:704-708).

[0025] Aloe is an intricate plant that contains many biologically activesubstances. (Cohen et al. in Wound Healing/Biochemical and ClinicalAspects, 1st ed. W B Saunders, Philadelphia (1992)). Over 300 species ofAloe are known, most of which are indigenous to Africa. Studies haveshown that the biologically active substances are located in threeseparate sections of the aloe leaf—a clear gel fillet located in thecenter of the leaf, in the leaf rind or cortex of the leaf and in ayellow fluid contained in the pericyclic cells of the vascular bundles,located between the leaf rind and the internal gel fillet, referred toas the latex. Historically, Aloe products have been used indermatological applications for the treatment of burns, sores and otherwounds. These uses have stimulated a great deal of research inidentifying compounds from Aloe plants that have clinical activity,especially anti-inflammatory activity. (See, e.g., Grindlay and Reynolds(1986) J. of Ethnopharmacology 16:117-151; Hart et al. (1988) J. ofEthnopharmacology 23:61-71). As a result of these studies there havebeen numerous reports of Aloe compounds having diverse biologicalactivities, including anti-tumor activity, anti-gastric ulcer,anti-diabetic, anti-tyrosinase activity (see, e.g., Yagi et al. (1977)Z. Naturforsch 32c:731-734) and antioxidant activity (Yu and Lee, U.S.Pat. No. 5,939,395).

[0026] Chromones isolated from various Aloe species have been reportedto have diverse biological activity. Aloesin (FIG. 2) reportedlyinhibits tyrosinase activity (Jones et al. Journal of Pigment CellResearch, Acceptance, Feb. 10^(th). 2002) and up-regulates cyclinE-dependent kinase activity (Lee et al. (1997) Biochem. Mol. Biol. Int.41:285-292). A c-glycosyl chromone isolated from Aloe barbadensisdemonstrates anti-inflammatory activity (Hutter et al. (1996) J. Nat.Prod. 59:541-543) and antioxidant activity similar to that ofalpha-tocopherol based on a rat brain homogenates model (Lee et al. FreeRadic Biol. Med. 28:261-265).

[0027]Aloe barbadensis leaves and its bitter principles exhibit effectson blood glucose level in normal and alloxan diabetic mice (Ajabnoor(1990) J. Ethnopharmacol. 28:215-220) and dried sap of various Aloespecies demonstrates anti-diabetic activity in clinical studies(Ghannam, (1986) Horm Res. 24:288-294).

[0028] Yagi et al. disclose a group of compounds isolated from Aloe,particularly aloesin and one of its derivatives, 2″-O-feruloylaloesin,which are effective inhibitors of tyrosinase. (Yagi et al. (1987) PlantMedica 515-517). Biochemical testing of the enzyme inhibition by meansof the Lineweaver Burk diagram showed that 2″-feruloylaloesin was anon-competitive inhibitor of tyrosinase, while aloesin is a competitiveinhibitor. Aloesin is a C-glucosylated 5-methylchromone (Holdsworth(1972) Chromones in Aloe Species, Part I-Aloesin PM 19(4):322-325). Invitro, aloesin is a strong inhibitor of tyrosinase activity (Yagi et al.(1987) Planta Medica 515-517). In assays of tyrosinase activity on thesubstrate L-DOPA, aloesin is capable of 50% inhibition at aconcentration of 0.2 mM.

[0029] U.S. Pat. No. 6,083,976, entitled “Method of Synthesis ofDerivatives of Aloesin,” describes a novel method for the synthesis ofderivatives of aloesin alkylated at the C-7 hydroxyl group. Thealkylated aloesins, produced by this method have the functionality ofaloesin, a tyrosinase-inhibiting compound with skin whitening activity,but have greater biological activity than aloesin as indicated by invitro tyrosinase assays. Additionally, the alkyl group makes thederivatized aloesins more fat soluble than aloesin, allowing them to beretained in the stratum corneum of the skin more effectively thanaloesin. As a result, the alkylated aloesins are more potent and fasteracting skin lightening agents than aloesin.

[0030] U.S. Pat. No. 6,123,959, entitled “Aqueous Composition ComprisingActive Ingredients for the De-Pigmentation of the Skin,” describesaqueous compositions comprising liposomes of phospholipids, and at leastone competitive inhibitor of an enzyme for the synthesis of melanin, incombination with at least one non-competitive inhibitor of an enzyme forthe synthesis of melanin. The competitive inhibitors of the inventioninclude aloesin and derivatives thereof. The invention also includes theuse of the compositions for the de-pigmentation of skin. Each of thesepatents is incorporated herein by reference in their entirety.

[0031] To date, known methods for purifying aloesin, as well as, otherchromones involve the use of chromatography. (See e.g., Rauwald and Beil(1993) J. of Chromatography 639:359-362; Rauwald and Beil (1993) Z.Naturforsch 48c:1-4; Conner et al. (1990) Phytochemistry 29:941;Holdsworth (1972) Chromones in Aloe Species, Part I-Aloesin PM19(4):322-325; Mebe (1987) Phytochemistry 26:2646; Haynes et al. (1970)J. Chem. Soc. (C) 2581; McCarthy and Haynes (1967) The Distribution ofAloesin in Some South African Aloe Species; Heft 3 342). Theseprocedures were developed for chemical analysis and are not practicalfor preparative scale production of aloesin. In U.S. patent applicationSer. No. 09/792,104, filed Feb. 26, 2001, entitled “Method ofPurification of Aloesin,” which is incorporated herein by reference inits entirety, a method for purification of aloesin using crystallizationis disclosed. Applicant knows of no report or suggestion of a method forthe purification of chromones from Aloe and other indicated speciesusing with polyamide or LH-20 column chromatography.

SUMMARY OF THE INVENTION

[0032] The present invention describes the identification andpurification of 7-hydroxychromes that exhibit potent antioxidantactivity. In one embodiment the present invention includes a method forproviding an antioxidant to a host in need thereof, comprisingadministering an effective amount of a 7-hydroxychrome or a mixture of7-hydroxychromones.

[0033] The present invention also includes methods that are effective ininhibiting lipid peroxidation through the simultaneous suppression offree radical generation and suppression of the production of reactiveoxygen species (ROS). The method for inhibiting lipid peroxidation iscomprised of administering a composition comprising a 7-hydroxychrome ora mixture of 7-hydroxychromones to a host in need thereof.

[0034] The present invention also includes methods for preventing andtreating ROS mediated diseases and conditions and diseases andconditions associated with other oxidative processes. The method forpreventing and treating ROS mediated diseases and conditions anddiseases and conditions associated with other oxidative processes iscomprised of administering to a host in need thereof an effective amountof a composition comprised of a 7-hydroxychrome or a mixture of7-hydroxychromones and a pharmaceutically acceptable carrier.

[0035] The 7-hydroxychromones that can be used in accordance with thefollowing include compounds illustrated by the following generalstructure:

[0036] wherein

[0037] R₁, R₂ and R₃ are independently selected from the groupconsisting of —H, —OH, —CH₃, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl,hydroxylalkyl, hydroxylalkenyl, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH,—NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, including,but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoylesters, trihydroxybenzoyl esters and caffeoyl esters; thereof carbon,oxygen, nitrogen or sulfur glycoside of a single or a combination ofmultiple sugars including, but not limited to, aldopentoses, methylaldopentose, aldohexoses, ketohexose and their chemical derivativesthereof; dimer, trimer and other polymerized chromones;

[0038] wherein said alkyl and/or alkenyl group is a straight and/orbranched chain having between 1-20 carbon atoms with and/or withoutdouble bonds in different positions;

[0039] X is selected from the group of pharmaceutically acceptablecounter anions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.; and

[0040] R is an alkyl group having between 1-20 carbon atoms. In apreferred embodiment of this invention the 7-hydroxychromone is selectedfrom aloesin (FIG. 2) or aloeresin A (FIG. 3).

[0041] The 7-hydroxychromones of this invention may be obtained bysynthetic methods or may be isolated from the genera of numerous plantfamilies, including but not limited to Acacia, Adina, Aloe, Alternaria,Amoora, Antidesma, Artemisia, Baeckia, Cassia, Clusea, Cnidium,Convolvulus, Epimedium, Eriosema, Eriostemon, Eugenia, Garcinia,Hypericum, Lindenbergia, Pancratium, Penicillium, Polygonum,Ptaeroxylon, Rheum, Sophora, Stephanitis, Syzygium, Talaromyces andZonaria. In a preferred embodiment, the plant is selected from thegroup, including but not limited to, Acacia catechu, Acacia concinna,Aloe arborescens, Aloe barbadensis, Aloe cremnophila, Aloe ferox, Aloesaponaria, Aloe vera, Aloe vera var. chinensis, Antidesma membranaceum,Artemisia capillaries, Baeckia frutescens, Epimedium sagittatum,Garcinia dulcis, Hypericum japonicum, Polygonum cuspidatum, Sophoratomentosa and Stephanitis rhododendri.

[0042] The 7-hydroxychromones can be found in different parts of plants,including but not limited to stems, stem barks, trunks, trunk barks,twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowersand other reproductive organs, leaves and other aerial parts.

[0043] The method of this invention can be used to treat and prevent anumber of ROS mediated diseases and conditions including, but notlimited to, atherosclerosis, coronary heart diseases, cataracts,dementia, Alzheimer's disease, cognitive dysfunction, diabetes mellitus,cancer, skin photo aging, skin wrinkles, sun burns, melanoma, and otherdegenerative processes associated with aging.

[0044] The compositions of this invention can be administered by anymethod known to one of ordinary skill in the art. The modes ofadministration include, but are not limited to, enteral (oral)administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application. The method oftreatment according to this invention comprises administering internallyor topically to a patient in need thereof a therapeutically effectiveamount of the individual and/or a mixture of multiple 7-hydroxychromones isolated from a single source or multiple sources thatinclude, but not limited to, synthetically obtained, naturallyoccurring, or any combination thereof.

[0045] The present invention also includes an improved method for theisolation and purification of chromones, particularly7-hydroxychromones, from plants containing these compounds. The methodof the present invention comprises: a) extracting the ground biomass ofa plant containing a chromone, particularly a 7-hydroxychromone; b)neutralizing and concentrating said extract; and c) purifying saidneutralized and concentrated extract using a chromatographic method,including but not limited to polyamide, LH-20 or reverse phasechromatography. In a preferred embodiment of the invention the extractis purified using a method selected from the group consisting ofrecrystallization, precipitation, solvent partition and/orchromatographic separation. The present invention provides acommercially viable process for the isolation and purification ofchromones having desirable physiological activity.

[0046] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

[0047]FIG. 1 depicts schematically a γ-Glutamyl Cycle.

[0048]FIG. 2 depicts the high-pressure liquid chromatography (HPLC)chromatogram of Aloesin.

[0049]FIG. 3 depicts the HPLC chromatogram of Aloeresin A.

[0050]FIG. 4 illustrates graphically the whole ORAC value of aloechromones Aloesin and Aloeresin A relative to grape seed extract andgreen tea extract.

[0051]FIG. 5 illustrates graphically the fast and slow anti-oxidantactivities of aloe chromones Aloesin and Aloeresin A relative to grapeseed extract and green tea extract.

[0052]FIG. 6 illustrates graphically the lipid peroxidation inhibitioncapacity (LPIC) of Aloeresin A relative to vitamin C andalpha-tocopherol.

[0053]FIG. 7 depicts graphically the improvement of ORAC value of ametaberry drink upon the addition of 1% and 5% Aloeresin A.

[0054]FIG. 8 depicts the improvement of lipid peroxidation inhibitioncapacity of an antioxidant formula upon the addition of Aloeresin A.

DETAILED DESCRIPTION OF THE INVENTION

[0055] Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

[0056] “Chromones” are a specific class of natural products having abenzopyran-4-one as the major structural skeleton as illustrated by thefollowing general structure:

[0057] wherein

[0058] R₁, R₂ and R₃ are independently selected from the groupconsisting of —H, —OH, —CH₃, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl,hydroxylalkyl, hydroxylalkenyl, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH,—NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, including,but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoylesters, trihydroxybenzoyl esters and caffeoyl esters; thereof carbon,oxygen, nitrogen or sulfur glycoside of a single or a combination ofmultiple sugars including, but not limited to, aldopentoses, methylaldopentose, aldohexoses, ketohexose and their chemical derivativesthereof; dimer, trimer and other polymerized chromones;

[0059] wherein said alkyl and/or alkenyl group is a straight and/orbranched chain having between 1-20 carbon atoms with and/or withoutdouble bonds in different positions;

[0060] X is selected from the group of pharmaceutically acceptablecounter anions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.; and

[0061] R is an alkyl group having between 1-20 carbon atoms. In apreferred embodiment of this invention the 7-hydroxychromone is selectedfrom aloesin (FIG. 1) or aloeresin A (FIG. 2).

[0062] The term “Aloe” refers to the genus of South African plants ofthe Liliaceae family of which the Aloe ferox is one of the species. Aloechromones are present primarily in the whole leaf of a number ofdifferent species of Aloe.

[0063] The term “Aloe extract” is defined as the dried juice of thewhole leaf of various species of the Aloe plant. The “Aloe extract” usedin the examples of this invention was prepared by “whole-leafprocessing” of the whole leaf of various Aloe species. In one example,whole leaves obtained from the Aloe barbadensis plant were ground,filtered, treated with cellulase (optional) and activated carbon andlyophilized. The lyophilized powder was reconstituted with thechromatography solvent prior to use. In another example, the exudatefrom aloe leaves was suspended in water, followed by contact with anappropriate chromatography solvent prior to use.

[0064] “Therapeutic” as used herein, includes treatment and/orprophylaxis. When used, therapeutic refers to humans, as well as, otheranimals.

[0065] “Pharmaceutically or therapeutically effective dose or amount”refers to a dosage level sufficient to induce a desired biologicalresult. That result may be the delivery of a pharmaceutical agent,alleviation of the signs, symptoms or causes of a disease or any otherdesirous alteration of a biological system.

[0066] A “host” is a living subject, human or animal, into which thecompositions described herein are administered.

[0067] Note, that throughout this application various citations areprovided. Each citation is specifically incorporated herein in itsentirety by reference.

[0068] The present invention describes the identification andpurification of 7-hydroxychromes that exhibit potent antioxidantactivity. In one embodiment the present invention includes a method forproviding an antioxidant to a host in need thereof, comprisingadministering an effective amount of a 7-hydroxychrome or a mixture of7-hydroxychromones.

[0069] The present invention also includes methods that are effective ininhibiting lipid peroxidation through the simultaneous suppression offree radical generation and suppression of the production of reactiveoxygen species (ROS). The method for inhibiting lipid peroxidation iscomprised of administering a composition comprising a 7-hydroxychrome ora mixture of 7-hydroxychromones to a host in need thereof.

[0070] The present invention also includes methods for preventing andtreating ROS mediated diseases and conditions and diseases andconditions associated with other oxidative processes. The method forpreventing and treating ROS mediated diseases and conditions anddiseases and conditions associated with other oxidative processes iscomprised of administering to a host in need thereof an effective amountof a composition comprised of a 7-hydroxychrome or a mixture of7-hydroxychromones and a pharmaceutically acceptable carrier.

[0071] The 7-hydroxychromones that can be used in accordance with thisinvention include compounds illustrated by the general structure setforth above. In a preferred embodiment of this invention the7-hydroxychromone is selected from aloesin (FIG. 1) or aloeresin A (FIG.2). The 7-hydroxychromones of this invention may be obtained bysynthetic methods or may be isolated from the genera of numerous plantfamilies, including but not limited to Acacia, Adina, Aloe, Alternaria,Amoora, Antidesma, Artemisia, Baeckia, Cassia, Clusea, Cnidium,Convolvulus, Epimedium, Eriosema, Eriostemon, Eugenia, Garcinia,Hypericum, Lindenbergia, Pancratium, Penicillium, Polygonum,Ptaeroxylon, Rheum, Sophora, Stephanitis, Syzygium, Talaromyces andZonaria. In a preferred embodiment, the plant is selected from thegroup, including but not limited to, Acacia catechu, Acacia concinna,Aloe arborescens, Aloe barbadensis, Aloe cremnophila, Aloe ferox, Aloesaponaria, Aloe vera, Aloe vera var. chinensis, Antidesma membranaceum,Artemisia capillaries, Baeckia frutescens, Epimedium sagittatum,Garcinia dulcis, Hypericum japonicum, Polygonum cuspidatum, Sophoratomentosa and Stephanitis rhododendri.

[0072] The 7-hydroxychromones can be found in different parts of plants,including but not limited to stems, stem barks, trunks, trunk barks,twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowersand other reproductive organs, leaves and other aerial parts.

[0073] Examples 1, 2 and 3 describe the isolation and purification oftwo representative 7-hydroxychromones, aloesin and aloeresin A. Aloesinand aloeresin A were isolated from the exudates of the leaves of Aloeferox in a purity of 95.2% and 61.5%, respectively.

[0074] As provided in Example 1, aloesin was isolated and purified usingthe methods described in U.S. patent application Ser. No. 09/792,104,filed Feb. 26, 2001, entitled “Method of Purification of Aloesin,” whichis incorporated herein by reference in its entirety. Aloesin can beobtained from Aloe exudates, such as aloe bitters or from other sources,including but not limited to, Aloe leaves, rinds, and whole plantscontaining this compound as a component.

[0075] The isolation and purification of aloesin A is described inExamples 2 and 3. In a typical separation, plant material containingaloeresin A (typically aloe bitters) is extracted in 10-100% alcohol (orother organic miscible solvent) in water (typically 30% methanol inwater). The aloesin A is then purified using typical chromatographicmedia, particularly polyamide or LH-20, and/or reversed phase media. Theextract solution is concentrated and/or directly applied to a bed ofpolyamide resin and/or a reversed phase based material. The column bedis then washed with 0-30% methanol (or other water soluble organicsolvent; typically 2 column volume of 30 % methanol in water) until allother compounds (typically aloesin and anthraquinones) are eluted. Thecolumn bed is then eluted with 30-100% organic solvent in water(typically two column volumes of 100% acetone). This fraction is thenconcentrated to obtain aloeresin A.

[0076] Example 4 describes the measurement of the Oxygen RadicalAbsorbance Capacity (ORAC) for both aloesin and aloeresin A, relative togrape seed extract and green tea extract. The results are set forth inFIG. 4. With reference to FIG. 4, it can be seen that aloesin had muchhigher whole ORAC value than aloeresin A and the standardized green teaand grape seed extracts. Aloeresin A had a whole ORAC value of 33 and299 higher than green tea and grape seed extracts, respectively. Example5 describes the measurement of fast and slow ORAC for the fourcompositions tested in Example 4. The results of this assay are setforth in FIG. 5. Aloeresin A was four times more potent as a fastantioxidant, than the other three compositions tested. Aloesin, however,was more potent as a slow acting antioxidant. Aloesin was over 70% moreactive than green tea extract and more than six times more active thangrape seed extract at 50% ORAC conditions. Aloeresin A was also asuperior antioxidant relative to grape seed and green tea extracts underthese conditions. Both aloesin and aloesin A contain phenolic hydroxylgroup in the C-7 position (see FIGS. 2 and 3). This hydroxyl group isvery easily oxidized by reactive oxygen species (ROS) to form a ketonegroup. The extra hydroxyl cinnamoyl group in aloeresin A does notenhance the whole ORAC value of this compound. However, it significantlychanges its polarity, which may be the reason that this compound is asuperior fast antioxidant.

[0077] Example 6 describes the evaluation of Lipid PeroxidationInhibition Capacity (LPIC) for aloeresin A, relative to vitamin C andα-tocopherol. As noted above, lipid peroxidation is believed to be amajor destructive reaction, occurring in the plasma components and bloodvessel walls and contributing to cardiovascular disease. The experimentdescribed in the Example 6, was designed as an indicator of free radicalinduced lipid destruction occurring in an organism. The LPIC assaymeasures the ability of a sample to both inhibit and terminate a lipidperoxidation reaction. The LPIC value complements the ORAC value. TheORAC assay provides information on total antioxidant capacity, whereasthe LPIC assay provides information on both total antioxidants and thestatus of metal catalysts in effectively protecting against in vivolipid peroxidation reactions.

[0078] The results of the assay described in Example 6 are set forth inFIG. 6. As illustrated in the FIG. 6, aloeresin A is a strongerantioxidant than either α-tocopherol or vitamin C, as measured by lipidperoxidation inhibition capacity (LPIC) from concentrations of 10 mg/mLto 50 mg/mL. It also inhibited lipid peroxidation 30.7% even at aconcentration as low as 5 mg/mL vs. below 10% for α-tocopherol.

[0079] Lipid peroxidation is controlled in vivo by chelation of tracemetals, such as, iron and copper that are involved in the initiation oflipid peroxidation reactions; and by the presence of antioxidants thatterminate the propagation reaction once it is initiated. Metalchelators, such as ferritin for iron and metallothionein for copper, arewell known, but other serum constituents, such as urate, which canchelate iron, may also be important. Antioxidants, such asalpha-tocopherol, are known to prevent propagation of lipid peroxidationreactions, but there may be many other serum constituents that could beequally or more important. Since aloeresin A contains a ketone, as wellas, a number of hydroxyl groups on the benzopyran-4-one ring and on thesugar unit, it is functional as both a metal chelator and a free radicalscavenger. Thus, a principal role of chromone antioxidants is theprotection of the entire cell system through the control of lipidperoxidation by both chelation and the scavenging of free radicals.

[0080] 7-hydroxychromones not only possess high ORAC and LPIC values inand of themselves, they can also improve the antioxidative capacity of aproduct if they are appropriately formulated into the product asillustrated in Examples 7 and 8. In the example 6, it is demonstratedthat aloeresin A, at a concentration of 5%, increases the ORAC value ofa multiple berry fruit drink by 31.3%. The results of Example 7 are setforth in FIG. 7. In Example 8, it is demonstrated that aloeresin Aincreases LIPC of a formulated antioxidant product by 19% to 38% at adosage of 50 mg to 250 mg/serving.

[0081] The method of this invention can be used to treat and prevent anumber of ROS mediated diseases and conditions including, but notlimited to, atherosclerosis, coronary heart diseases, cataracts,dementia, Alzheimer's disease, cognitive dysfunction, diabetes mellitus,cancer, skin photo aging, skin wrinkles, sun burns, melanoma, and otherdegenerative processes associated with aging.

[0082] The compositions of this invention can be administered by anymethod known to one of ordinary skill in the art. The modes ofadministration include, but are not limited to, enteral (oral)administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application. The method oftreatment according to this invention comprises administering internallyor topically to a patient in need thereof a therapeutically effectiveamount of the individual and/or a mixture of multiple 7-hydroxychromones isolated from a single source or multiple sources thatinclude, but not limited to, synthetically obtained, naturallyoccurring, or any combination thereof.

[0083] The present invention also includes an improved method for theisolation and purification of chromones, particularly7-hydroxychromones, from plants containing these compounds. The methodof the present invention comprises: a) extracting the ground biomass ofa plant containing a chromone, particularly a 7-hydroxychromone; b)neutralizing and concentrating said extract; and c) purifying saidneutralized and concentrated extract using a chromatographic method,including but not limited to polyamide, LH-20 or reverse phasechromatography. In a preferred embodiment of the invention the extractis purified using a method selected from the group consisting ofrecrystallization, precipitation, solvent partition and/orchromatographic separation. The present invention provides acommercially viable process for the isolation and purification ofchromones having desirable physiological activity.

[0084] The preparation of products for administration in pharmaceuticalpreparations may be performed by a variety of methods well known tothose skilled in the art. The 7-hydroxychromones may be formulated as anherb powder in the form of their natural existence; as solvent and/orsupercritical fluid extracts in different concentrations; as enrichedand purified compounds through recrystallization, column separation,solvent partition, precipitation and other means, as a pure and/or amixture containing substantially purified 7-hydroxychromones prepared bysynthetic methods.

[0085] Various delivery systems are known in the art and can be used toadminister the therapeutic compositions of the invention, e.g., aqueoussolution, encapsulation in liposomes, microparticles, and microcapsules.

[0086] Therapeutic compositions of the invention may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally andtopically active formulations, transdermal iontophoresis orsuppositories are also envisioned. One preferred carrier isphysiological saline solution, but it is contemplated that otherpharmaceutically acceptable carriers may also be used. In one preferredembodiment, it is envisioned that the carrier and 7-hydroxychromone(s)constitute a physiologically compatible, slow release formulation. Theprimary solvent in such a carrier may be either aqueous or non-aqueousin nature. In addition, the carrier may contain other pharmacologicallyacceptable excipients for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, rate of dissolution, orodor of the formulation. Similarly, the carrier may contain still otherpharmacologically acceptable excipients for modifying or maintaining thestability, rate of dissolution, release or absorption of the ligand.Such excipients are those substances usually and customarily employed toformulate dosages for parental administration in either unit dose ormulti-dose form.

[0087] Once the therapeutic composition has been formulated, it may bestored in sterile vials as a solution, suspension, cream, gel, emulsion,solid, or dehydrated or lyophilized powder. Such formulations may bestored either in a ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing the compositions for systemic delivery may bevia subcutaneous, intramuscular, intravenous, topical, intranasal orvaginal or rectal suppository.

[0088] The amount of the composition that will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder of condition, which can be determined by standardclinical techniques. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness or advancement of thedisease or condition, and should be decided according to thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. For example, an effective amount of the compositionof the invention is readily determined by administering graded doses ofthe composition and observing the desired effect.

[0089] The method of treatment according to this invention comprisesadministering internally or topically to a patient in need thereof atherapeutically effective amount of the individual and/or a mixture of7-hydroxychromones from a single source or multiple sources. The purityof the individual and/or a mixture of 7-hydroxychromones includes, butis not limited to 0.01% to 100%, depending on the methodology used toobtain the compound(s). In a preferred embodiment doses of the7-hydroxychrome and pharmaceutical compositions containing the same arean efficacious, nontoxic quantity generally selected from the range of0.01 to 200 mg/kg of body weight. Persons skilled in the art usingroutine clinical testing are able to determine optimum doses for theparticular ailment being treated.

[0090] This invention includes an improved method for isolating andpurifying 7-hydroxychromones from plants. The improved method of thisinvention comprises: extraction of the ground biomass of a plantcontaining 7-hydroxychromone with single or combination of organicsolvent and/or water; neutralization and concentration of theneutralized extract; and purification of said extract by polyamide orLH-20 chromatography. As provided above, these 7-hydroxychromones can beidentified from the genera of numerous plant families. The method ofthis invention can be extended to the isolation of these compounds fromany plant source containing these compounds.

[0091] Additionally the 7-hydroxychromones can be isolated from variousparts of the plant including, but not limited to, the whole plant,stems, stem bark, twigs, tubers, flowers, fruit, roots, root barks,young shoots, seeds, rhizomes and aerial parts. In a preferredembodiment the 7-hydroxychromones are isolated from the whole leaves ofAloe ferox.

[0092] The following examples are provided for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLES Example 1

[0093] Preparation of Aloesin from the Exudates of Aloe ferox

[0094] A sample of aloesin was prepared using the methods described U.S.patent application Ser. No. 09/792,104, filed Feb. 26, 2001, entitled“Method of Purification of Aloesin,” which is incorporated herein byreference in its entirety. Briefly, Aloe extract was isolated from thewhole leaf of Aloe ferox that had been previously dissolved in hot waterand filtered to remove undissolved particulates. The extract was thenloaded onto a reverse phase column and aloesin was eluted from thecolumn with 20-30% methanol.

[0095] Preparative HPLC

[0096] Column: IB SIL C18, 250 mm×4.6 mm, 5μ particle size

[0097] Mobile Phase: water/methanol gradient: 80%/20% (20 minutes);40%/60% (10 minutes); 80%/20% (10 minutes)

[0098] Temperature: Ambient

[0099] Flow Rate: 1 mL/min

[0100] Detector Wavelength: 297

[0101] Sensitivity: 20

[0102] Product eluted between 8-9 minutes

[0103] The compound was further purified by recrystalization to yield alight yellow solid with >95.2% purity.

Example 2

[0104] Preparation of Aloeresin A from the Exudates of Aloe ferox

[0105] A mixture of aloe exudates (35 kg) isolated from Aloe ferox, asdescribed in U.S. patent application Ser. No. 09/792,104, filed Feb. 26,2001, entitled “Method of Purification of Aloesin,” which isincorporated herein by reference in its entirety, was added to hot water(200 L at 74° C.) and stirred for 1 hour. The solution was allowed tostand at room temperature for 2 days. The aqueous layer was decanted andthe thick liquid residue was extracted again with hot water (200 L at60° C.). A portion of this secondary extract (20 L) was concentrated andevaporated to dryness.

[0106] Dry extract (1.5 kg), containing 20.1% of aloeresin A, wasdissolved in 25 L DI water. This solution was stirred for 30 minutes andloaded directly onto a pre-prepared polyamide column. The pre-preparedcolumn (20 cm×100 cm) contained 20 L of polyamide and was cleaned withaqueous sodium hydroxide and then acidified with aqueous HCl. The columnwas then washed with DI water to neutral condition before the loading ofthe solution containing aloeresin A.

[0107] The load material was fed into the bottom of the column at about1.5 L per minute keeping the pressure under 10 psi. The column waswashed with water (20 L) followed by a methanol/water mixture (20 L 25%methanol). The column was blown dry using a peristaltic pump. AloeresinA was then eluted with acetone (20L). The eluent was collected, analyzedand evaporated to yield total of 377 g of a solid that contained 61.5%Aloeresin A.

[0108] Example 3

[0109] Purification of Aloeresin A by LH-20 Column Chromatography

[0110] A dry extract (9.6 g), containing approximately 45% of aloeresinA, was dissolved in 250 mL DI water. This solution was loaded directlyonto a pre-prepared LH-20 column. The pre-prepared column (6.0 cm×12.0cm) contained 400 mL of LH-20 resin and was equilibrated with 2 columnvolumes of DI water. The load material was fed into the top of thecolumn and eluted with 800 mL 30% methanol followed by 600 mL of 100%methanol. The column was then equilibrated with 800 mL DI water andready to be used again. A total of 38 fractions were collected with 50mL in each fraction. Fractions 1-14 contained no compounds and werediscarded. Fractions 15-30 were combined and evaporated to provide 1.43g of anthraquinones in total solid of 4.615 g. The Aloeresin A was infractions 31-36. These fractions were combined and evaporated to yield4.05 g of Aloeresin A with a purity of 98%. The total solid recoveryfrom the LH-20 column chromatography was 90.3%.

Example 4

[0111] Measurement of the Oxygen Radical Absorption Capacity (ORAC) ofAloeresin A and Aloesin

[0112] The chromones, Aloesin and Aloeresin A, were tested for OxygenRadical Absorption Capacity (ORAC) relative to green tea extract whichcontains more than 50% total catechins and more than 25%epigallocatechingallate (EGCG) and grape seed extract which containsmore than 95% total phenols. The ORAC assay measures the time-dependentdecrease in the fluorescence of β-phycoerythrin (β-PE) indicatorprotein, resulting from oxygen radical damage. This assay provides aquantitative measure of the total antioxidant capacity to protect thefree radical sensitive β-phycoerythrin. Each reaction was calibratedusing the known standard Trolox, a water soluble vitamin E analogue. TheORAC value refers to the net protection area under the quenching curveof β-phycoerythrin in the presence of an antioxidant. The results of theassay are reported on the basis of 1 ORAC unit equals 1 μM of Trolox.Aloeresin A (purity 76.0%), aloesin (95.2%), green tea extract and grapeseed extract were dissolved in deionized water (5% solution W/V) andtested directly using the experimental procedures described in Cao etal. (1994) Free Radic. Biol. Med. 16:135-137 and Prior and Cao (1999)Proc. Soc. Exp. Biol. Med. 220:255-261. The results are set forth in theFIG. 4.

Example 5

[0113] Quantification of Fast and Slow Oxygen Radical AbsorptionCapacity (ORAC) for Aloeresin A and Aloesin

[0114] The fast antioxidant activity is a measure of the net protectionarea under the quenching curve of β-phycoerythrin in the presence of anantioxidant, which completely inhibits free radical propagation, to theextent that 95% of β-phycoerythrin survives. Ascorbic acid, freephenolic compounds and thiol compounds are fast acting antioxidants. Theslow antioxidant activity is a measure of the net protection area underthe quenching curve of β-phycoerythrin in the presence of anantioxidant, which inhibits free radical propagation to the extent that50% of β-phycoerythrin survives. Flavonoids, carotenoids and polyphenolsare slow acting antioxidants. Aloeresin A (purity 76.0%), Aloesin(95.2%), green tea extract and grape seed extract, respectively weredissolved in deionized water (5% (W/V) solution) and tested directlyusing the experiment procedures described by Cao et al. (1994) FreeRadic. Biol. Med. 16:135-137 and Prior and Cao (1999) Proc. Soc. Exp.Biol. Med. 220:255-261. The results are set forth in FIG. 5.

Example 6

[0115] Quantification of Lipid Peroxidation Inhibition Capacity (LPIC)of Aloeresin A Relative to Vitamin C and alpha-Tocopherol

[0116] Lipid Peroxidation Inhibition Capacity (LPIC) is a measure of theanti-oxidative ability of a sample to inhibit the initiation andpropagation of a spontaneous lipid peroxidation reaction.Malonyldialdehyde (MDA) is one of the major by-products of lipidperoxidation and is commonly used as a means of measuring the extent oflipid peroxidation. MDA concentrations were measured using thethiobarbituric acid reaction. Aloeresin A (purity 76.0%), vitamin C andα-tocopherol were dissolved in a phosphate-saline buffer at fourdifferent concentrations (5 mg/mL, 10 mg/mL, 25 mg/mL and 50 mg/mL) andthen tested directly using the experiment procedures described by Stockset al. (1974) Clinical Sci. Mol. 47:215-222 and Yu and Lee, U.S. Pat.No. 5,939,395. The results are set forth in FIG. 6.

Example 7

[0117] Improvement of Oxygen Radical Absorption Capacity (ORAC) for aMeta-Berry Drink Product by Addition of Aloeresin A

[0118] A fruit drink product obtained from Oasis Wellness Network wasdetermined, using the methods described in Example 3, to have an ORACvalue of 3,129/oz. Aloeresin A, containing 76.0% aloeresin A was addedto 40 mL of the drink (lot #0612, exp. 11-02) to produce a finalconcentration of 5 mg/mL and 15 mg/mL aloeresin A in the solution,respectively. These samples were then tested for whole ORAC valuerelative to the product in the absence of aloeresin A. using theexperiment procedures illustrated described by Cao et al. (1994) FreeRadic. Biol. Med. 16:135-137 and Prior and Cao (1999) Proc. Soc. Exp.Biol. Med. 220:255-261). The results are set forth in the FIG. 7.

Example 8

[0119] Improvement of Lipid Peroxidation Inhibition Capacity (LPIC) foran Anti-Oxidant Product by Addition of Aloeresin A

[0120] An anti-oxidant product in a formulated tablet form obtained fromOasis Wellness Network was ground and mixed with a sample of aloeresin A(76.0% purity) in a final concentration equivalent to 50 mg/tablet and250 mg/tablet, respectively. These samples were then tested for LipidPeroxidation Inhibition Capacity (LPIC), as compared to the productabsent aloeresin A, using the experiment procedures described by Stockset al. (1974) Clinical Sci. Mol. 47:215-222 and Yu and Lee, U.S. Pat.No. 5,939,395. The test results are illustrated in the FIG. 8.

What is claimed is
 1. A method for providing an antioxidant to a host inneed thereof, comprising administering an effective amount of a7-hydroxychrome or a mixture of 7-hydroxychromones.
 2. The method ofclaim 1 wherein said 7-hydroxychrome is selected from the group ofcompounds having the following structure:

wherein R₁, R₂ and R₃ are independently selected from the groupconsisting of —H, —OH, —CH₃, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl,hydroxylalkyl, hydroxylalkenyl, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH,—NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, selectedfrom the group consisting of gallate, acetate, cinnamoyl andhydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters;thereof carbon, oxygen, nitrogen or sulfur glycoside of a single or acombination of multiple sugars selected from the group consisting ofaldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedchromones; wherein said alkyl and/or alkenyl group is a straight and/orbranched chain having between 1-20 carbon atoms with and/or withoutdouble bonds in different positions; X is selected from the group ofpharmaceutically acceptable counter anions including hydroxyl, chloride,iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.; and R isan alkyl group having between 1-20 carbon atoms.
 3. The method of claim2 wherein said 7-hydroxychrome is selected from aloesin or aloeresin A.4. The method of claim 1 wherein said 7-hydroxychrome is obtained byorganic synthesis.
 5. The method of claim 1 wherein said 7-hydroxychromeis obtained from a plant part.
 6. The method of claim 5 wherein saidplant is selected from the group consisting of the genus of Acacia,Adina, Aloe, Alternaria, Amoora, Antidesma, Artemisia, Baeckia, Cassia,Clusea, Cnidium, Convolvulus, Epimedium, Eriosema, Eriostemon, Eugenia,Garcinia, Hypericum, Lindenbergia, Pancratium, Penicillium, Polygonum,Ptaeroxylon, Rheum, Sophora, Stephanitis, Syzygium, Talaromyces andZonaria.
 7. The method of claim 5 wherein said plant is selected fromthe group consisting of Acacia catechu, Acacia concinna, Aloearborescens, Aloe barbadensis, Aloe cremnophila, Aloe ferox, Aloesaponaria, Aloe vera, Aloe vera var. chinensis, Antidesma membranaceum,Artemisia capillaries, Baeckia frutescens, Epimedium sagittatum,Garcinia dulcis, Hypericum japonicum, Polygonum cuspidatum, Sophoratomentosa, and Stephanitis rhododendri.
 8. The method of claim 5 whereinsaid plant part is selected from the group consisting of stems, stembarks, trunks, trunk barks, twigs, tubers, roots, root barks, youngshoots, seeds, rhizomes, flowers and other reproductive organs, leavesand other aerial parts.
 9. The method of claim 1, wherein saidantioxidant acts through the simultaneous suppression of free radicalgeneration and suppression of the production of reactive oxygen species(ROS).
 10. The method of claim 9 wherein said 7-hydroxychrome isselected from the group of compounds having the following structure:

wherein R₁, R₂ and R₃ are independently selected from the groupconsisting of —H, —OH, —CH₃, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl,hydroxylalkyl, hydroxylalkenyl, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH,—NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, selectedfrom the group consisting of gallate, acetate, cinnamoyl andhydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters;thereof carbon, oxygen, nitrogen or sulfur glycoside of a single or acombination of multiple sugars selected from the group consisting ofaldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedchromones; wherein said alkyl and/or alkenyl group is a straight and/orbranched chain having between 1-20 carbon atoms with and/or withoutdouble bonds in different positions; X is selected from the group ofpharmaceutically acceptable counter anions including hydroxyl, chloride,iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.; and R isan alkyl group having between 1-20 carbon atoms.
 11. The method of claim10 wherein said 7-hydroxychrome is selected from aloesin or aloeresin A.12. The method of claim 9 wherein said 7-hydroxychrome is obtained byorganic synthesis.
 13. The method of claim 9 wherein said7-hydroxychrome is obtained from a plant part.
 14. The method of claim13 wherein said plant is selected from the group consisting of the genusof Acacia, Adina, Aloe, Alternaria, Amoora, Antidesma, Artemisia,Baeckia, Cassia, Clusea, Cnidium, Convolvulus, Epimedium, Eriosema,Eriostemon, Eugenia, Garcinia, Hypericum, Lindenbergia, Pancratium,Penicillium, Polygonum, Ptaeroxylon, Rheum, Sophora, Stephanitis,Syzygium, Talaromyces and Zonaria.
 15. The method of claim 13 whereinsaid plant is selected from the group consisting of Acacia catechu,Acacia concinna, Aloe arborescens, Aloe barbadensis, Aloe cremnophila,Aloe ferox, Aloe saponaria, Aloe vera, Aloe vera var. chinensis,Antidesma membranaceum, Artemisia capillaries, Baeckia frutescens,Epimedium sagittatum, Garcinia dulcis, Hypericum japonicum, Polygonumcuspidatum, Sophora tomentosa, and Stephanitis rhododendri.
 16. Themethod of claim 13 wherein said plant part is selected from the groupconsisting of stems, stem barks, trunks, trunk barks, twigs, tubers,roots, root barks, young shoots, seeds, rhizomes, flowers and otherreproductive organs, leaves and other aerial parts.
 17. A method forpreventing and treating ROS mediated diseases and conditions comprisingadministering to a host in need thereof an effective amount of acomposition having a 7-hydroxychrome or a mixture of 7-hydroxychromonesand a pharmaceutically acceptable carrier.
 18. The method of claim 17wherein said 7-hydroxychrome is selected from the group of compoundshaving the following structure:

wherein R₁, R₂ and R₃ are independently selected from the groupconsisting of —H, —OH, —CH₃, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl,hydroxylalkyl, hydroxylalkenyl, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH,—NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, selectedfrom the group consisting of gallate, acetate, cinnamoyl andhydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters;thereof carbon, oxygen, nitrogen or sulfur glycoside of a single or acombination of multiple sugars selected from the group consisting ofaldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedchromones; wherein said alkyl and/or alkenyl group is a straight and/orbranched chain having between 1-20 carbon atoms with and/or withoutdouble bonds in different positions; X is selected from the group ofpharmaceutically acceptable counter anions including hydroxyl, chloride,iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.; and R isan alkyl group having between 1-20 carbon atoms and a pharmaceuticallyacceptable carrier.
 19. The method of claim 18 wherein said7-hydroxychrome is selected from aloesin or aloeresin A.
 20. The methodof claim 17 wherein said compound is obtained by organic synthesis. 21.The method of claim 17 wherein said compound is from a plant part. 22.The method of claim 21 wherein said plant is selected from the groupconsisting of the genus of Acacia, Adina, Aloe, Alternaria, Amoora,Antidesma, Artemisia, Baeckia, Cassia, Clusea, Cnidium, Convolvulus,Epimedium, Eriosema, Eriostemon, Eugenia, Garcinia, Hypericum,Lindenbergia, Pancratium, Penicillium, Polygonum, Ptaeroxylon, Rheum,Sophora, Stephanitis, Syzygium, Talaromyces and Zonaria.
 23. The methodof claim 21 wherein said plant is selected from the group consisting ofAcacia catechu, Acacia concinna, Aloe arborescens, Aloe barbadensis,Aloe cremnophila, Aloe ferox, Aloe saponaria, Aloe vera, Aloe vera var.chinensis, Antidesma membranaceum, Artemisia capillaries, Baeckiafrutescens, Epimedium sagittatum, Garcinia dulcis, Hypericum japonicum,Polygonum cuspidatum, Sophora tomentosa, and Stephanitis rhododendri.24. The method of claim 21 wherein said plant part is selected from thegroup consisting of stems, stem barks, trunks, trunk barks, twigs,tubers, roots, root barks, young shoots, seeds, rhizomes, flowers andother reproductive organs, leaves and other aerial parts.
 25. The methodof claim 17 wherein the ROS mediated diseases and conditions areselected from the group consisting of atherosclerosis, coronary heartdiseases, cataracts, dementia, Alzheimer's disease, cognitivedysfunction, diabetes mellitus, cancer, skin photo aging, skin wrinkles,sun burns, melanoma, and other degenerative processes associated withaging.
 26. The method of claim 17 wherein the composition is comprisedof 0.01% to 100% of the 7-hydroxychrome.
 27. The method of claim 17wherein the composition is administered in a dosage selected from 0.01to 200 mg/kg of body weight.
 28. The method of claim 17 wherein theroutes of the administration are selected from the group consisting oforal, topical, suppository, intravenous, and intradermic, intragaster,intramusclar, intraperitoneal and intravenous administration in anappropriate pharmaceutical formula.
 29. A method for isolating andpurifying a composition of 7-hydroxychromones from plants containingsaid compounds, said method comprising: a) extracting the ground biomassof the plant with a solvent; b) neutralizing and concentrating saidextract; and c) purifying said extract from step b) by polyamide orLH-20 chromatography.
 30. The method of claim 29 wherein the compositionof chromones is extracted with a solvent selected from the groupconsisting of water, alcohol, an organic solvent and/or a combinationthereof.
 31. The method of claim 29 wherein the extract is furtherpurified using a method selected from the group consisting ofrecrystallization, solvent partition, precipitation, sublimation, and/orchromatographic separation.